Phage display vector, phages encoded thereby, and methods of use thereof

ABSTRACT

Filamentous bacteriophages are particularly efficient for the expression and display of combinatorial random peptides. Two phage proteins are often employed for peptide display: the infectivity protein, pIII and the major coat protein, pVIII. The use of pVIII typically requires the expression of two pVIII genes: the wild type and the recombinant pVIII genes to generate mosaic phages. “Type 88” vectors contain two pVIII genes in one phage genome. A novel “type 88” expression vector has been rationally designed and constructed, which can be used to express recombinant peptides as pVIII chimeric proteins in mosaic bacteriophages. This vector is not only genetically stable but also of high copy number and produces high titers of recombinant phages.

FIELD OF THE INVENTION

The present invention is related to a phage display vector encoding afilamentous phage which includes DNA encoding a polypeptide of interestand, more particularly, to such a vector in which a recombinant sequenceincluding a multiple cloning site is inserted between the wild typepVIII and pIII genes. The invention further relates to the phagesencoded by such DNA, phage display libraries made of such phages andmethods of use thereof.

BACKGROUND OF THE INVENTION

Combinatorial phage display peptide libraries provide an effective meansto study protein:protein interactions. This technology relies on theproduction of very large collections of random peptides associated withtheir corresponding genetic blueprints (Scott et al, 1990; Dower, 1992;Lane et al, 1993; Cortese et al, 1994; Cortese et al, 1995; Cortese etal, 1996). Presentation of the random peptides is often accomplished byconstructing chimeric proteins expressed on the outer surface offilamentous bacteriophages such as M13, fd and f1. This presentationmakes the repertoires amenable to binding assays and specializedscreening schemes (referred to as biopanning (Parmley et al, 1988))leading to the affinity isolation and identification of peptides withdesired binding properties. In this way peptides that bind to receptors(Koivunen et al, 1995; Wrighton et al, 1996; Sparks et all, 1994;Rasqualini et al, 1996), enzymes (Matthews et al, 1993; Schmitz et al,1996) or antibodies (Scott et al, 1990; Cwirla et al, 1990; Felici etal, 1991; Luzzago et al, 1993; Hoess et al, 1993; Bonnycastle et al,1996) have been efficiently selected.

Filamentous bacteriophages are nonlytic, male specific bacteriophagesthat infect Escherichia coli cells carrying an F-episome (for review,see Model et al, 1988). Filamentous phage particles appear as thintubular structures 900 nm long and 10 nm thick containing a circularsingle stranded DNA genome (the +strand). The life cycle of the phageentails binding of the phage to the F-pilus of the bacterium followed byentry of the single stranded DNA genome into the host. The circularsingle stranded DNA is recognized by the host replication machinery andthe synthesis of the complementary second DNA strand is initiated at thephage ori(−) structure. The double stranded DNA replicating form is thetemplate for the synthesis of single-stranded DNA circular phagegenomes, initiating at the ori(+) structure. These are ultimatelypackaged into virions and the phage particles are extruded from thebacterium without causing lysis or apparent damage to the host.

Peptide display systems have exploited two structural proteins of thephage; pIII protein and pVIII protein. The pIII protein exists in 5copies per phage and is found exclusively at one tip of the virion(Goldsmith et al, 1977). The N-terminal domain of the pIII protein formsa knob-like structure that is required for the infectivity process (Grayet al, 1981). It enables the adsorption of the phage to the tip of theF-pilus and subsequently the penetration and translocation of the singlestranded phage DNA into the bacterial host cell (Holliger et al, 1997).The pIII protein can tolerate extensive modifications and thus has beenused to express peptides at its N-terminus. The foreign peptides havebeen up to 65 amino acid residues long (Bluthner et al, 1996; Kay et al,1993) and in some instances even as large as full-length proteins(McCafferty et al, 1990; McCafferty et al, 1992) without markedlyaffecting pIII function.

The cylindrical protein envelope surrounding the single stranded phageDNA is composed of 2700 copies of the major coat protein, pVIII, anα-helical subunit which consists of 50 amino acid residues. The pVIIIproteins themselves are arranged in a helical pattern, with the α-helixof the protein oriented at a shallow angle to the long axis of thevirion (Marvin et al, 1994). The primary structure of this proteincontains three separate domains: (1) the N-terminal part, enriched withacidic amino acids and exposed to the outside environment; (2) a centralhydrophobic domain responsible for: (i) subunit:subunit interactions inthe phage particle and (ii) transmembrane functions in the host cell;and (3) the third domain containing basic amino acids, clustered at theC-terminus, which is buried in the interior of the phage and isassociated with the phage-DNA. pVIII is synthesized as a precoat proteincontaining a 23 amino acid leader-peptide, which is cleaved upontranslocation across the inner membrane of the bacterium to yield themature 50-residue transmembrane protein (Sugimoto et al, 1977). Use ofpVIII as a display scaffold is hindered by the fact that it can toleratethe addition of peptides no longer than 6 residues at its N-terminus(Greenwood et al, 1991; Iannolo et al, 1995). Larger inserts interferewith phage assembly. Introduction of larger peptides, however, ispossible in systems where mosaic phages are produced by in vivo mixingthe recombinant, peptide-containing, pVIII proteins with wild type pVIII(Felici et al, 1991; Greenwood et al, 1991; Willis et al, 1993). Thisenables the incorporation of the chimeric pVIII proteins at low density(tens to hundreds of copies per particle) on the phage surfaceinterspersed with wild type coat proteins during the assembly of phageparticles. Two systems have been used that enable the generation ofmosaic phages; the “type 8+8” and “type 88” systems as designated bySmith (Smith, 1993).

The “type 8+8” system is based on having the two pVIII genes situatedseparately in two different genetic units (Felici et al, 1991; Greenwoodet al, 1991; Willis et al, 1993). The recombinant pVIII gene is locatedon a phagemid, a plasmid that contains, in addition to its own origin ofreplication, the phage origins of replication and packaging signal. Thewild type pVIII protein is supplied by superinfecting phagemid-harboringbacteria with a helper phage. In addition, the helper phage provides thephage replication and assembly machinery that package both the phagemidand the helper genomes into virions. Therefore, two types of particlesare secreted by such bacteria, helper and phagemid, both of whichincorporate a mixture of recombinant and wild type pVIII proteins.

The “type 88” system benefits by containing the two pVIII genes in oneand the same infectious phage genome. Thus, this obviates the need for ahelper phage and superinfection. Furthermore, only one type of mosaicphage is produced. The question arises, however, where one shouldintroduce the second pVIII gene within the filamentous phage genome forefficient expression and genetic stability.

The phage genome encodes 10 proteins (pI through pX) all of which areessential for production of infectious progeny (Felici et al, 1991). Thegenes for the proteins are organized in two tightly packedtranscriptional units separated by two non-coding regions (Van Wezenbeeket al, 1980). One non-coding region, called the “intergenic region”(defined as situated between the pIV and pII genes) contains the (+) andthe (−) origins of DNA replication and the packaging signal of thephage, enabling the initiation of capsid formation. Parts of thisintergenic region are dispensable (Kim et al, 1981; Dotto et al, 1984).Moreover, this region has been found to be able to tolerate theinsertion of foreign DNAs at several sites (Messing, 1983; Moses et al,1980; Zacher et al, 1980). The second non-coding region of the phage islocated between the pVIII and pIII genes, and has also been used toincorporate foreign recombinant genes as was illustrated by Pluckthun(Krebber et al, 1995).

Regardless as to where a second pVIII gene is to be introduced, a majorpoint for concern is the genetic stability of the ultimate vector andits derivatives.

SUMMARY OF THE INVENTION

The present invention is based on a critical examination of theattributes of the two non-coding regions of the fd filamentous phage aspotential sites for insertion of a second recombinant pVIII gene and itsgenetic stability, resulting in the design and construction of anefficient “type 88” phage display expression system.

The phage display expression system of the present invention includes avector which is the DNA sequence of a filamentous phage into which asecond pVIII gene, as well as DNA encoding a peptide of interest, areplaced between the wild type pVIII and pIII genes. This allowsproduction of type 88 phages displaying a peptide of interest withgenetic stability and high copy number.

The DNA encoding the second pVIII gene preferably uses alternativecodons for encoding the amino acid residues of the pVIII protein. Thenative pVIII DNA sequence is separated from the recombinant pVIIIsequence by a region encoding the wild type pVIII C-terminal domain anda terminator. The region encoding the wild type pVIII C-terminal domainis preferably designed to use alternative codons which are other thanthe native codons for encoding the same amino acids. In this wayhomologous recombination, slippage mechanisms and other geneticinstabilities may be avoided.

Preferably, the terminator at the end of the native pVIII DNA sequenceis an HP terminator which is not native to the filamentous phage DNA.

In a preferred embodiment of the present invention, a positive selectionmarker, such as the tetracycline or kanamycin resistant genes, isinserted between the native and recombinant pVIII genes. Preferably, aunidirectional promoter is substituted for the native bidirectionaltetracycline resistant gene promoter.

In preferred embodiments of the present invention, the native intergenicspace between pIV and pII is maintained.

The present invention further relates to the filamentous phages encodedby the DNA discussed above. Such phages may be of any type, such as fd,M13 and f1, although fd is preferred. The peptide designed to bedisplayed by the phage of the present invention can be any peptide whichis desired to be presented, such as a specific antigen. Indeed, anyprotein or peptide can be displayed on the phage of the presentinvention.

The peptide displayed on the phage may be an epitope of an antigen,which phage can be used therapeutically as a vaccine. Furthermore, thepeptide displayed on the phage may be a single-chain antibody, whichphage can be used for passive immunization. Both of these embodimentsare described in WO 01/18169.

Alternatively, one can create a library of phages into which areincorporated, at the same site, each of a set of oligonucleotides thatencode all possible random peptides of a given length, or a large subsetof that set. Such a large subset should preferably represent a fractionof the full set with the theoretical complexity of the full set ofrandom polypeptides of a given length.

Libraries of phages can also be prepared which incorporate all, or alarge subset of all, of the overlapping oligonucleotides that representall of the overlapping peptides of a given antigen and, thus, create aphage display pepscan. Scrambled pepscans (PCT application no. WO98/20169) and phage display two hybrid systems (PCT application no. WO98/20159) may also be prepared by this technique.

The phage display libraries of the present invention can be used inscreening for molecules which bind to a particular displayed peptide ofinterest or in screening the peptides displayed on a library of phagesto see which bind to a specific molecule of interest, all as is wellknown with respect to prior art phage display libraries. Once a moleculeor peptide of interest is identified by means of the screen using thephages or phage display libraries of the present invention, the peptideor molecule so identified may be produced in a conventional manner.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are a comparison between fd (FIG. 1A) and fd-tet (FIG.1B). Opened arrows indicate the phage-transcriptional units. Thedarkened segment in FIG. 1B indicates the tet fragment introduced intothe BamH I site of fd at position 6272 (bold in FIG. 1A) in theintergenic region (IG).

FIGS. 2A-2B show a deletion of 2.8 kb in fd-tet. FIG. 2A is an uncutdouble-stranded DNA of fd-tet isolated from K802 (lane 1) or K91KAN(lane 2) and run on 0.8% agarose gel. Uncut double-stranded DNA of wildtype fd was used for comparison (lane 3). M: DNA ladder mix (numbersequal base pairs). FIG. 2B shows Msc I digests of fd-tet derived fromK802 (lane 1) or K91KAN (lane 2) which were run on 0.8% agarose gel. MscI digest of fd was used for comparison (lane 3). M: λ Hind III digest(numbers equal base pairs). Note that the lower band in lane 2 in FIG.2B, corresponding to the deleted product, is at least as intense as theupper band corresponding to the linear full-length fd-tet .

FIG. 3 shows the genetic instability of the inserted tet fragment. SnaBI/Ava I digests of fd-tet derived from K802 (lane 1) or K91KAN (lane 2).SnaB I/Ava I digest of fd was used for comparison (lane 3). BspH I/HindIII digests of fd-tet derived from K802 (lane 4) or K91KAN (lane 5:).BspH I/Hind III digest of fd DNA was used for comparison (lane 6). Thearrow indicates the deleted products (lane 2 and 5) corresponding insize to the linear full-length fd (lane 3 and 6). M: DNA ladder mix.Agarose gel concentration was 0.8%.

FIGS. 4A-4B show that the deletion is not a precise reversion to fd.FIG. 4A is BamH I digests of fd-tet DNA derived from K802 (lane 1) orK91KAN (and 2). BamH I digest of fd was used for comparison (lane 3).Note that the pattern obtained in lane 2 is identical to the Msc Idigest in FIG. 2B, lane 2. M: DNA ladder mix. Agarose gel concentrationwas 0.8%. FIG. 4B is BstY I digests of fd-tet derived from K802 (lane 1)or K91KAN (lane 2) run on 0.8% agarose gel. BstY I digest of fd was usedfor comparison (lane 3). M: DNA ladder mix. Note the relative drop inintensity for the lowest band in FIG. 4B, lane 2.

FIG. 5 shows the genome of the fd-tet phage. Genes (darkened segments),promoters (bent arrows), terminators (stem and loop), and selectedrestriction sites relevant to this study are shown. Strong promoters andterminators are in black. Weak promoters and terminators are in gray.The wild type fd genes (pI through pX) are packed in two transcriptionalunits. The inserted tet fragment contains two genes; tetA encoding forthe tetracycline resistance protein and tetR encoding for the repressorprotein regulating the expression of the tetA gene. The overlappingpromoter/terminator, between pVIII and pIII genes, are inseparable.Moreover, the −35 box of this promoter is situated in the C-terminalcoding region of pVIII gene (see text). Note the bi-directional promoterdriving tetR and teta transcription.

FIGS. 6A-6C show the construction of two tandem pVIII genes. FIG. 6A isa detailed comparison between the wild type pVIII gene (the contiguouscentral portion being SEQ ID NO:1) and the recombinant pVIII gene (thecontiguous central portion being SEQ ID NO:3), designated pVIIISTS. Theamino acid sequence shown to be encoded by SEQ ID NO:1 is SEQ ID NO:2.The amino acid sequences shown to be encoded by SEQ ID NO:3 are SEQ IDNOs:4 and 5. In the pVIII gene, the GAC codon for Asp at position 4 ofthe wild type pVIII is deleted and replaced by a sequence of 62 bpcontaining two stop codons and trpA transcription terminator flanked bytwo Sfi I sites (“STS” insert) (nucleotides 13-74 of SEQ ID NO:3). FIG.6B is the pGEM-t(p8STS) construct. The fragment containing the pVIIISTSgene, preceded by a SnaB I restriction site and followed by downstreampIII gene, was introduced into the pGEM-T vector. Black segments aresequences corresponding to the pGEM-T vector. The 62 bp “STS” insert isindicated. FIG. 6C is the pGEM-t(pB8STS) construct. This constructcontains the wild type pVIII and the modified pVIIISTS genes arranged astandem repeats. The fragment containing the wild type pVIII genestarting from the SnaB I site and ending just beyond the overlappingpromoter/terminator was introduced into the SnaB I site of pGEM-T(p8STS)thus destroying the former site while concomitantly introducing a newSnaB I site upstream to the wild type pVIII gene. Furthermore,additional unique sites were introduced between the two pVIII genes.

FIG. 7 is the genome of ftac88 vector. Most of the tetR gene wasexchanged for a linker containing the Ava II site, thus eliminating thesecond SnaB I site previously situated in this region (for comparisonsee FIG. 5). The modified pVIIISTS gene is situated between theduplicated overlapping promoter/terminator structures, withoutdisrupting the two-phage transcriptional units. Note the pVIIISTS geneis under the control of a tac promoter (Ptac).

FIG. 8 shows the binding of mAb GV4H3 to phages expressing the GV4H3epitope. Equal amounts of phages were applied in duplicates to anitrocellulose membrane filter and reacted with the GV4H3 mAb. The GV4H3epitope displaying phages, ftac88(4H3) and fth1(4H3), showed strongsignals. Phage ftac88 and fth1 were used as negative controls.

FIG. 9 shows the deletion of the recombinant pVIII gene in ftac88(4H3).SnaB I/BamH I digests of ftac88 derived from DH5α (lane 1) or DH5αF′(lane 2) were run on 0.8% agarose gel. SnaB I/BamH I digests of ftac88(4H3) isolated from DH5α (lane 3) or DH5αF′ (lane 4) are also shown. M:DNA ladder mix. The upper arrow indicates the tet insert deleted productand the lower arrow indicates the recombinant pVIII gene deletedfragment.

FIG. 10 shows the fth1 vector (SEQ ID NO:29). The general scheme of thefth1 vector is shown in scale illustrating the phage genes (openedsegments), the reconstituted intergenic region (IG) and the genesintroduced between the wild type pVIII and pIII genes (gray segments).Below, detail of the pIX to the middle of pIII is provided (not inscale). The darkened segments represent the modified wild type pVIII3′-end and the −35 box of the pIII promoter (see also FIG. 11). Fordetails see text.

FIGS. 11A-11C show the intermediate vectors constructed for theformation of fth1 vector. FIG. 11A is a detail of the segment of fd-tetcontaining the pIX, pVIII, and pIII genes. The segment in pVIIIindicating the −35 box of the pIII promoter is shown in black situatedin the 3′ region of the wild type pVIII open reading frame. FIG. 11B isthe intermediate vector-1 (IV-1). The −35 box of the pIII promoter wasseparated from the wild type pVIII gene by inserting a small segmentreconstituting the 3′ region and introducing multiple cloning sites.FIG. 11C is IV-4. A novel HP terminator (t_(HP)) is situated downstreamto the wild type pVIII gene terminating its transcription followed bythe tetracycline resistance gene (tetA) under the control of the kanpromoter (P_(kan)).

FIG. 12 shows the genetic stability of fth1. BstN I digests of fth1derived from DH5α (lane 2) or DH5αF′ (lane 3). BstN I digests offth1(4H3) derived from DH5α (lane 4) or DH5αF′ (lane 5). BstN I digestof fd was used for comparison (lane 1). M: DNA ladder mix. Agarose gelconcentration was 0.8%.

DETAILED DESCRIPTION OF THE INVENTION

Type 88 display systems are in essence a phage genome that contains twoprotein VIII genes. One of the genes is the wild type pVIII, whereas thesecond is modified so as to enable expression of a peptide (random orpre-defined peptide, or even a single-chain antibody (scFv)). Theproblem with known type 88 phage display systems is genetic instability.After a number of generations, the phages tend to stop production of theinserted polypeptide of interest and no longer display such polypeptide.The novel constructs of the present invention solve this problem.Furthermore, in prior art systems, in which the selectable marker hasbeen introduced into the intergenic region, some of the functions of thereplication of the phage are obstructed, and the actual amount of phageDNA being produced in a liter of culture may be no more than 20-50 μg ofDNA. However, with the construct of the present invention, high copynumbers may be obtained. Thus, as much as 500-1000 μg of DNA can bereadily purified from a liter of culture.

In the vector of the present invention, the sequence of a filamentousphage is manipulated so as to insert a second pVIII gene and the geneencoding the polypeptide of interest between the wild type pVIII andpIII genes. In a preferred embodiment, the intergenic region between pIIand pIV is not modified and retains the native sequence, thus allowingfor the high copy number to be obtained. Any positive selection markermay also be introduced between the wild type pVIII and pIII genes.

In the wild type filamentous phage DNA, the −35 box necessary for pIIIexpression is positioned to overlap with the C-terminal coding region ofthe pVIII protein. Thus, the −35 regulatory sequence is upstream of theC-terminal coding region and terminator of the pVIII protein. So as notto disrupt the expression of the pIII protein, the vector of the presentinvention preferably maintains the genuine C-terminal/−35 box at the 5′end of the pIII gene. A new C-terminal region for the native pVIII geneis introduced upstream thereof. The second pVIII region and the geneencoding the polypeptide of interest is introduced between thenewly-introduced terminator for the native pVIII gene and theC-terminal/−35 box 5′ to the pIII gene.

In order to prevent possible homologous recombination, slippagemechanisms, or other genetic instabilities, it is preferable that theDNA encoding the second pVIII gene use alternative codons which areother than the native codons for encoding amino acid residues of thepVIII protein. Enough alternative codons should be inserted therein soas to avoid stretches longer than 30 bases of the native sequence,preferably no longer than 20 such bases. Similarly, the region encodingthe wild type pVIII C-terminal domain should also use alternative codonsin sufficient quantity to avoid stretches longer than 30 native bases,and preferably to avoid stretches longer than 20 such bases.

The positive selection marker is preferably inserted between the nativeand recombinant pVIII genes.

The present invention further comprehends a vector in which the secondpVIII gene is introduced just downstream to the −35 promotor of the pIIIgene (and thus driven by the pIII promotor and not the tac promotor offth1 or ftac88 as described). In such a situation, due to the presenceof the transcription terminator in the STS stuffer of the recombinantpVIII, phages that do not incorporate an insert for the polypeptide ofinterest will not be produced at all. This is due to the fact that theterminator prevents the expression of the pIII, pVI and pI proteins. ThepI protein is essential for phage assembly. In the situation where aninsert is cloned in place of the stuffer, the recombinant phage is made.If such an insert is not incorporated, then transcription is terminatedand no phage is produced.

While the fd filamentous phage is used in the present examples and isthe preferred phage sequence for use in the present invention, it shouldbe understood that all filamentous phages are very similar and have thesame gene organization (Model et al, 1988). Thus, the principles of thepresent invention can be applied to any of the filamentous phages, suchas M13, f1 and others.

The native pVIII DNA sequence is preferably separated from the secondpVIII DNA sequence by a terminator which is not native to thefilamentous phage DNA, preferably the HP terminator.

In the preferred embodiment of the present invention, the native,unique, overlapping pVIII terminator/pIII promoter is maintained at itsnative position at the N-terminal portion of the pIII gene. Thisincludes the pVIII C-terminal region/pIII −35 box. However, to furtheravoid tandem repeats, the pVIII C-terminal region, which is present atthis point, is preferably designed with alternative codon usage to avoidstretches of longer than 30 native bases, and preferably to avoidstretches longer than 20 native bases.

While the DNA molecule of the present invention is described as having arecombinant DNA sequence encoding the pVIII protein and DNA encoding apolypeptide of interest other than pVIII which is inserted between thewild type pVIII and pIII genes, this language encompasses the presenceof DNA encoding a peptide of interest within the sequence encoding thepVIII protein. Preferably, the DNA sequence encoding the peptide ofinterest is present at the N-terminal region of the pVIII gene. Thepeptide of interest and the pVIII gene will be expressed as a fusionprotein in which the peptide of interest is expressed on the surface ofthe filamentous phage.

While the selectable marker disclosed herein is the tetracyclineresistance gene, those of ordinary skill in the art will understand thatany selectable marker can be used for this purpose, such as thekanamycin resistance gene. Furthermore, as the native promoter for thetetracycline resistance gene is bidirectional, in a preferred embodimentof the present invention, this promoter is replaced by a unidirectionalpromoter, such as the kanamycin promoter.

The present invention also comprehends a vector which may be used as anintermediate for producing a vector containing the DNA encoding thepeptide of interest. In this intermediate vector, the DNA of the phagehas inserted therein a recombinant DNA sequence that includes a multiplecloning site. The multiple cloning site has a series of restrictionsites that do not otherwise appear in the DNA of the phage into whichthe recombinant DNA has been inserted. The recombinant DNA sequence isdesigned and inserted such that the multiple cloning site is placedbetween a terminator for the wild type pVIII gene and an initiator forthe wild type pII gene. The multiple cloning site may then be used toreadily insert DNA sequences encoding the foreign peptide of interest tobe displayed on the phage. A positive selection marker, as has beendiscussed elsewhere in the present specification, may be present in themultiple cloning site in order to simplify insertion only of the DNAencoding the peptide of interest so as to arrive at the final product.The second pVIII gene may also be present in the multiple cloning site.

Instead of a second pVIII gene, any recombinant bacteriophage gene canbe present in the multiple cloning site. For example, it would be usefulto produce a system in which two pIII genes are present. This wouldallow the production of both wild type and recombinant pIII genes in thesame phage. It is expected that, by modifying the efficiency ofexpression, one can create a situation in which only one recombinantpIII protein is expressed per phage. The normal situation is that fivepIII proteins are present per phage. When all are recombinant, then thebinding is affected by the multivalency of the five copies which leadsto enhanced avidity of binding and can be misleading if one wants to beprecise in evaluating real affinity (as opposed to avidity whichbenefits from multiple binding reactions). Similarly, any of the nativeproteins (pI through pX) can be duplicated in the multiple cloning sitefor whatever reason.

The recombinant DNA sequence, which is inserted into the DNA of thefilamentous phage, is designed and inserted such that the multiplecloning site is placed between a terminator for the wild type pVIII geneand an initiator for the wild type pIII gene. As discussed hereinabove,preferably the wild type C-terminal/−35 box is retained at the 5′ end ofthe pIII gene, and a new C-terminal region for the native pVIII gene isintroduced upstream of the multiple cloning site.

Preferably, in the intermediate vector, in the region between flankingrestriction enzyme sites, a trpA transcription terminator is disposed,although any random combination of bases can appear between theseflanking restriction enzyme sites. When this intermediate is to be usedfor production of the vector to be used to make a phage displaying thepeptide of interest, DNA encoding the peptide of interest is merelysubstituted for the DNA of the intermediate vector between the twoflanking unique restriction enzyme sites.

Since the recombinant DNA sequence inserted into the phage in order tocreate the multiple cloning site must include a region homologous to theN-terminal portion of the pIII gene, it is possible in this same insertto include a DNA sequence encoding another foreign peptide incorporatedinto the pIII gene at the N-terminal region thereof. This may be used,for example, to produce a phage that displays a library of randompeptides on either pIII or pVIII and a marker on the other, such asgreen fluorescence protein. This would make the selection of the boundphage easier or amenable to high throughput screening.

Also encompassed by the present invention is the recombinant DNAconstruct which is produced and used for insertion of the multiplecloning sites, into the phage DNA so as to appear between the pVIII andpIII genes. This construct is designed so as to replace the wild typesequence between restriction sites which are already present in thephage, such as the SnaBI in the pIX gene and the BamHI in the pIII gene.The ends of the construct are designed so as to be compatible with therestriction enzyme cleavage sites left after removal of the wild typesequence such that efficient ligation of the construct into the vectormay be obtained, all as is well known to those of ordinary skill in theart.

The novel DNA vectors of the present invention and the novel type 88filamentous phages encoded thereby may be used in the same manner thatknown peptide display phages have been known to be used in the priorart, except that they are genetically stable, have high copy number, andproduce high titres of phages. The vector can be used to express anyprotein or peptide. In the situation in which a discrete sequence ofpeptide is known which is desired to be expressed, then such a knownpeptide can readily be incorporated into the vector so as to producehigh titres of phage displaying the desired discrete amino acidsequence.

An example of the use of a discrete sequence of peptide displayed on aphage is represented by the disclosure of PCT application no. WO01/18169, the entire contents of which are hereby incorporated herein byreference. This publication discloses the utility as a vaccine of phagesdisplaying a particular antigenic epitope so as to raise antibodiesagainst that epitope. Also disclosed therein are phages displayingsingle-chain antibodies which can be directly used for passiveimmunization. Thus, phages displaying such antigens or single-chainantibodies for use in the particular applications of WO 01/18169, or anyrelated application, are considered to be comprehended by the presentinvention when the antigen or single-chain antibody is displayed on aphage in accordance with the present invention.

Additionally, as is known in the art, libraries can be prepared thatincorporate into the same site of the vector a set of oligonucleotidesthat code all possible random peptides of a given length, or a largesubset of that set, as is known in the art. Such a large subset shouldpreferably represent a fraction of the full set with the theoreticalcomplexity of the full set of random polypeptides of a given length.Such phage display libraries can be used to screen for amino acidcombinations which will bind to a given target molecule. Once a phagewhich binds to the desired target molecule is found, the peptide insertin that phage can be determined and molecules containing that peptidecan be produced. Such molecule would be expected to bind the targetmolecule. The binding of the target molecule to its native receptor orligand may be disrupted. This is useful when it is desired to preventthe signaling which occurs when the target molecule is bound to itsnative ligand or receptor, all as is well known in the art of phagedisplay libraries.

Furthermore, libraries can be prepared which incorporate the set of allof the overlapping oligonucleotides that represent all the overlappingpeptides of a given antigen, or a large subset thereof, and, thus,create a phage display pepscan. A scrambled pepscan, such as thecombinatorial scrambled vaccines described in PCT application no. WO98/20169, the entire contents of which are hereby incorporated herein byreference, and the phage display two hybrid systems, such as aredescribed in PCT application no. WO 98/20159, the entire contents ofwhich are hereby incorporated by reference, may also be prepared usingthe novel phages of the present invention.

Any of such phage display libraries can be used in screen assays as isknown in the art. In any such screening assay, the peptide that is foundcan then be produced and used for its intended purpose, again, as iswell known in the prior art. An example of such a screen would be to usea library in accordance with the present invention against a receptor.In this way one might be able to select for a peptide that mimics thenative ligand. This peptide could then be produced using standardMerrifield synthesis, and the synthetic peptide may be used as either alead peptide for further development or directly as a modulator of thereceptor being studied. For example, a random phage display peptidelibrary may be used to screen against HIV gp120. In this way, one mightbe able to select for a peptide that binds to gp120 at precisely the CD4binding site. Such a peptide would be expected to bind to the virus andto prevent the association of the virus to the CD4 receptor and, thus,prevent infection. One could also produce a library derived from CD4itself and hope to discover a fragment of CD4 that binds to gp120 andacts as a decoy. All possibilities stem from the fact that the libraryis produced in the genetically stable expression system of the presentinvention.

As to the selectable marker, a particular advantage of placing thismarker between the two pVIII genes is to create a situation that if, forwhatever reason, there is a genetic recombination based on the limitedhomology of the native pVIII gene and the recombinant pVIII gene, thenthe selectable marker would be excised and lost as well. Running anexperiment in the presence of the antibiotic would result in loss ofthose recombinants that have lost their resistance gene. Other markersthat could be used in place of the tetracycline resistance gene would bethe ampicillin resistance gene, β-lactamase or chloramphenicolresistance gene.

As stated hereinabove, it is also possible within the scope of thepresent invention to also insert an exogenous gene between the pIII geneand its promoter. By doing so, the phage can display a library of randompeptides on either pIII or pVIII and a marker on the other, such asgreen fluorescence protein. This would make the selection of the boundphage easier or amenable to high throughput screening. The new exogenousgene can either be inserted directly between the pIII gene and itspromoter or a second pIII gene can be inserted similarly to thatdiscussed in the present invention for the second pVIII gene in order toensure that the native pIII gene will always be produced along with thesecond pIII gene and the peptide of interest. It is not necessary toinsert a separate pIII gene as, in contrast to pVIII, pIII can toleratelarge inserts.

The following examples are directed to preferred embodiments inaccordance with the present invention and show how to make and use thepresent invention. Those of ordinary skill in the art will understand,however, that these examples do not detract from the breadth of thepresent invention as described herein and that appropriate substitutionscan be made without engaging in undue experimentation by those ofordinary skill in the art.

Materials and Methods

Bacterial Strains, Phages, Reagents, and General Techniques

E. coli strains used in this study were the following. K802: F⁻e14⁻(McrA⁻) lacY1 or Δ(lac)6 supE44 galK2 galT22 rfbD1 metB1 mcrB1hsdS3(r_(K) ⁻ m_(K) ⁻). K91KAN: a derivative of K91 (Hfr-Cavalli, thi)in which the “mini-Kan hopper” element was inserted in the lacZ gene ofK91 rendering this strain kanamycin resistant (Parmley et al, 1988).DH5α: F⁻ endA1 hsdR17(r_(K) ⁻ m_(K) ⁺) supE44 thi-1 recA1 gyrA(Nal^(r))relA1 Δ(lacIZYA-argF)U169 deoR (Φ80dlacΔ(lacZ)M15). DH5αF′: F′ endA1hsdR17(r_(K) ⁻ m_(K) ⁺) supE44 thi-1 recA1 gyrA(Nal^(r)) relA1Δ(lacIZYA-argF)U169 deoR (Φ80dlacΔ(1acZ)M15). JM109: F′[traD36 lacI^(q)Δ(lacZ)M15 proA⁺B⁺] e14⁻(McrA⁻) Δ(lac-proAB) thi gyrA96(Nal^(r)) endA1hsdR17(r_(K) ⁻ m_(K) ⁺) relA1 supE44 recA1. XL1-Blue: F′[::Tn10 proA⁺B⁺lacI^(q) Δ(lacZ)M15] recA1 endA1 gyrA96(Nal^(r)) thi hsdR17(r_(K) ⁻m_(K) ⁺) supE44 relA1 lac. MC1061: F⁻ araD139 Δ(ara-leu)7696 galE15galK16 Δ(lac)X74 rpsL(Str^(r)) hsdR2(r_(K) ⁻ m_(K) ⁺) mcrA mcrB1. NM554:F⁻ araD139 Δ(ara-leu)7696 galE15 galK16 Δ(lac)X74 rpsL(Str^(r))hsdR2(r_(K) ⁻ m_(K) ⁺) mcrA mcrB1 recA13.

The wild type fd filamentous phage and fd-tet vector were kindlyprovided by G. P. Smith (University of Missouri, Columbia, Mo.). TheM13K07 phage was purchased from New England BioLabs Inc., MA (NEB). DNAwas isolated using the alkaline lysis procedure and purified on a cesiumchloride gradient as described previously (Stern et al, 1998). All therestriction enzymes used were purchased from NEB and the digestions wereperformed following the manufacturer's instructions. The monoclonalantibody GV4H3 was produced from a Balb/c mouse immunized with the HIV-1envelope protein gp120 (Denisova et al, 1996).

Oligonucleotides

The oligonucleotides used in this study were the following. ON1:5′-GCCTTCGTAGTGGCATTACG-3′ (SEQ ID NO: 6) ON2:5′-AGCCCGCTCATTAGGCGGGCTTCATTACCGGCCACGTCGGCCAC (SEQ ID NO: 7)CCTCAGCAGCGAAAGAC-3′ ON3:5′-CGGTAATGAAGCCCGCCTAATGAGCGGGCTTTTTTTTGGCCTCT (SEQ ID NO: 8)GGGGCCGATCCCGCAAAAGCGGCC-3′ ON4: 5′-GGTCAGACGATTGGCCTTG-3′ (SEQ ID NO:9) ON5: 5′-GAGCTCGCTAGCGCTCGAGCCAAAAAAAAAGGCTCCAAAAGG-3′ (SEQ ID NO: 10)ON6: 5′-CTAGAGCAGGGTCCAGCTAA-3′ (SEQ ID NO: 11) ON7: 5′-CTGGACCCTGCT-3′(SEQ ID NO: 12) ON8: 5′-TCGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAA(SEQ ID NO: 13) TTGTGAGCGGATAACAATTGAGCT-3′ ON9:5′-CAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGATG (SEQ ID NO: 14)ATTAATTGTCAACAGC-3′ ON10: 5′-CCGTGCATCTGTCCTCGTTC-3′ (SEQ ID NO: 15)ON11: 5′-CAGGCTTAAGCATCGACGTCTTATCAAGACGCCTTGCTTGTAA (SEQ ID NO: 16)ACTTTTTGAATAACTTGATACCGATAGTTGCGC-3′ ON12:5′-GTCTTGATAAGACGTCGATGCTTAAGCCTGGCTAGCCATCAGAT (SEQ ID NO: 17)CTGAGTCGGCCGCTGTTTAAGAAATTCACCTCG-3′ ON13:5′-GCCGTACCGCTAGCATTAGAAAAACTCATCGAGC-3′ (SEQ ID NO: 18) ON14:5′-GCTTCCTGACAGGAGGCCGTTTTGTTTTGCAGCCCACCTGAGCT (SEQ ID NO: 19)CCCAGCTTAAGGTGTCTCAAAATCTCTGATG-3′ ON15:5′-GGCTGAGGGACGTCGAGGGCATGCGTACCCGATAAAAGCGGCTT (SEQ ID NO: 20)CCTGACAGGAGGCCG-3′ ON16: 5′-AGCCTTTCAGGTCAGAAGGG-3′ (SEQ ID NO: 21)ON17: 5′-ATTCATGCCGGAGAGGGTAG-3′ (SEQ ID NO: 22) ON18:5′-CATAACACCCCTTGTATTACTG-3′ (SEQ ID NO: 23) ON19:5′-GCTTACATAAACAGTAATACAAGGGGTGTTATGAATAGTTCGAC (SEQ ID NO: 24)AAAGATCGC-3′ ON20: 5′-GGATCGAGAGCTAGCATAACTAAGCACTTGTCTCCTG-3′ (SEQ IDNO: 25) ON21: 5′-TGCCGGCGCTCCCGCTGGTTTCGCTATCCTGTCAGCACCCGGGT (SEQ IDNO: 26) CTG-3′ ON22: 5′-ACCCGGGTGCTGACAGGATAGCGAAACCAGCGGGAGCGCCGGCA(SEQ ID NO: 27) CGT-3′Vector Construction

In this study two novel vector-systems were constructed. The rationalefor their compositions and structures is described in the Results.Detailed diagrams of the vectors and sub-structures are given in theFigures as indicated. The following are the details of the specificsteps taken to construct the vectors.

Construction of ftac88

The ftac88 vector (for detailed map see FIG. 7) was constructed throughthe following steps. First, the recombinant pVIII gene, designatedpVIIISTS (for orientation see FIG. 6A), was generated by introducing a62 bp insert three codons downstream to the leader peptide using the“SOEing” PCR mutagenesis (Horton et al, 1990). For this, fouroligonucleotides were used. ON1 and ON2 were used to PCR amplify from fda 169 bp fragment. ON3 and ON4 were used to PCR generate from fd a 899bp fragment. ON2 and ON3 each contain 5′-extension corresponding to the62 bp insert. Thus, the resulting PCR fragments contain an identical 30bp stretch of the novel sequence. The two fragments were purified fromagarose gel, mixed and used as a combined template for PCR amplificationusing the oligonucleotides ON1 and ON4 to generate the final 1071 bpproduct containing the modified pVIII gene, part of pIII gene, and theflanking BamH I-SnaB I sites. The PCR reaction was performed by usingthe Taq polymerase which adds adenine to the 3′ ends of PCR products andthus the resulting 1071 bp fragment was directly ligated with thelinearized pGEM-T vector (Promega Corporation, WI) containingcomplementary 3′ thymidine overhangs, generating the pGEM-T(p8STS)construct (see FIG. 6B).

Next, the wild type pVIII gene was PCR amplified from fd using theoligonucleotides ON1 and ON5. ON5 has a 5′ extension that contains therestriction sites Xho I, Nhe I and Sac I. The PCR reaction was performedusing the Pwo Polymerase (Boehringer Mannheim), resulting in blunt-ended335 bp fragment from the SnaB I site to just beyond the overlappingpromoter/terminator followed by the ON5 introduced restriction sites.The resulting fragment was ligated with the blunt-ended SnaB Ilinearized pGEM-T(p8STS) construct generating the pGEM-T(p88STS)construct (see FIG. 6C).

To remove the SnaB I site in tetR gene, the fd-tet vector (for detailedmap see FIG. 5) was digested with Xba I and BstX I restriction enzymesand the 8458 bp fragment was purified from agarose gel. The purifiedfragment was ligated with a linker containing the Ava II site producedby annealing the two complementary oligonucleotides ON6 and ON7.

To exchange the SnaB I-BamH 1952 bp segment of fd-tet for the SnaBI-BamH 11378 bp segment of pGEM-T(p88STS) construct containing both thewild type and recombinant pVIII genes, the Ava II containing fd-tetderivative was digested with SnaB I/BamH I and the 7519 bp fragment waspurified from agarose gel. In the same manner, the pGEM-T(p88STS)construct was digested with SnaB I/BamH I and the released 1378 bpfragment was purified from agarose gel. These two fragments were ligatedto generate an intermediate vector.

This intermediate vector was digested with Sac I and Xho I restrictionenzymes and the 13 bp stuffer was removed by applying the DNA digest ona chroma-spin™ column (Clontech Laboratories, Inc., Palo Alto, Calif.).The linearized intermediate vector was ligated with a linker containingthe tac promoter produced by annealing the two complementaryoligonucleotides ON8 and ON9, generating the ftac88 vector.

Construction of fth1

The construction of the fth1 vector (for detailed map see FIG. 10) wasperformed through a series of intermediate vectors. The intermediatevector-1 (IV-1, see FIG. 11B) was constructed by introducing a DNAsegment ten codons upstream to the stop codon of the wild type pVIIIgene using the “SOEing” PCR mutagenesis described above. Two fragmentsof 519 bp and 827 bp were PCR generated from fd using theoligonucleotides ON10/ON11 and ON12/ON4 respectively. The two fragmentswere mixed and used as a combined template using ON10/ON4 to PCRgenerate the final 1316 bp fragment. This product was digested with SnaBI and BamH I and the resulting 1034 bp fragment was purified fromagarose gel and used to replace the corresponding SnaB I-BamH I fragmentof the Ava II containing fd-tet derivative described above for theconstruction of ftac88.

Next, the IV-2 was constructed by introducing into the IV-1, downstreamto the wild type pVIII gene, the novel HP transcription terminatorfollowed by the kanamycin resistance gene. This was performed in order(i) to functionally separate the two phage-transcriptional units and(ii) to enable the reconstitution of the intergenic region whilemaintaining an alternative antibiotic selectable marker (i.e.kanamycin). The kanamycin resistance gene was PCR amplified from M13K07using the oligonucleotides ON13 and ON14. Both oligonucleotides contain5′ extensions enabling the incorporation of additional flankingsequences. The 3′ addition contained the Nhe I restriction site and the5′ extension contained additional cloning sites adjacent to thekanamycin promoter and part of the HP terminator. The resulting 944 bpfragment was purified from agarose gel and used as a template for PCRamplification using the oligonucleotides ON13 and ON15. The latter has a5′ extension containing the second part of the HP terminator andadditional cloning sites ending with the Aat II site. Thus the resulting984 bp fragment contains the entire HP terminator bracketed betweenmultiple cloning sites followed by the kanamycin resistance gene. Thisproduct was digested with Nhe I and Aat II and the resulting fragmentwas purified from agarose gel. The purified fragment was ligated withNhe I/Aat II digested IV-1 vector after removing the 20 bp stuffer byapplying the vector digest on a chroma-spin™ column, ultimatelygenerating IV-2.

The IV-2 was used to reconstitute the intergenic region containing theorigins of replication. This was performed by PCR amplifying theintergenic region from fd phage using the oligonucleotides ON16 andON17. The resulting 1081 bp fragment containing the Msc I-Drd I segmentwas digested with Msc I and Drd I restriction enzymes. The 679 bp MscI-Drd I product was purified from agarose gel and used to exchange theMsc I-Drd I fragment in IV-2 containing the tet fragment, generatingIV-3.

The open reading frame only of the kanamycin resistance gene wasreplaced by the open reading frame of the tetracycline resistance genethus constructing the IV-4 vector (see FIG. 11C). This was performedusing the “SOEing” PCR. The oligonucleotides ON1 and ON18 were used toPCR amplify from IV-2 the 463 bp fragment containing the upstreamsequence to the ATG initiating codon of the kanamycin gene. Theoligonucleotides ON19 and ON20 were used to PCR amplify from fd-tet the1265 bp fragment containing the open reading frame of the tetracyclineresistance gene. The two fragments were mixed and used for PCRamplification of the 1698 bp fragment using the oligonucleotides ON1 andON20. The PCR was designed to flank the resulting 1698 bp fragmentbetween Nhe I and Sac I restriction sites enabling the digestion of thisfragment with the corresponding enzymes. After purifying the digestedfragment from agarose gel, it was used to replace the corresponding NheI-Sac I fragment of IV-3.

The final step was to generate the synthetic recombinant pVIII geneusing a panel of overlapping oligonucleotides (not shown). Part of thepanel was used as templates and the other part as 5′-extended primersfor PCR amplification thus generating separately small pieces of DNA.These small fragments were sequentially mixed and used as a combinedtemplate for the “SOEing” PCR ultimately generating the recombinantpVIIISTSh gene that subsequently was introduced between the Bgl II/Eag Isites of IV-4 producing the fth1 vector.

Introduction of the GV4H3 Epitope into ftac88 and fth1 Vectors

The vectors ftac88 and fth1 were digested with Sfi I restriction enzymeand the 49 bp stuffer containing the trpA terminator (for orientationsee FIG. 6 a) was removed by applying the DNA digest on a chroma-spin™column. The purified linear vector was ligated with GV4H3 epitopeencoding linker produced by annealing the two complementaryoligonucleotides ON21 and ON22.

Dot Blot Analysis of GV4H3 Epitope Presenting Phages

The phages were applied via a vacuum manifold to a nitrocellulosemembrane filter. After blocking (5% evaporated spray dried skim milk1.5% fat in Tris buffered saline (TBS)) for 1 hour, the membrane waswashed briefly with TBS and incubated overnight with 1 μg/ml of GV4H3mAb in TBS/5% milk at 4° C. with gentle rocking. After washing, themembrane was incubated with goat-anti mouse IgG/HRP conjugate diluted1:5000 in TBS/5% milk for 1 hour at room temperature. The positivesignals were detected by ECL (Amersham International plc,Buckinghamshire, England) immunodetection.

Results

The fd-tet vector of Smith (Zacher et al, 1980) was used as a startingpoint for our studies.

The fd-tet Expression Vector

Using the fd genome as a scaffold molecule, Zacher et al (1980)constructed the fd-tet phage, which contains the tetracycline resistancegene as a selectable marker (see FIG. 1). The fragment coding for thetetracycline resistance was obtained from the transposon Tn10 (a 2775 bpfragment flanked between two Bgl II sites). This segment was introducedinto the BamH I site of the intergenic region containing the origins ofreplication of the fd genome. The fd-tet vector has the followingadvantages:

-   -   1. fd-tet phages confer selectable tetracycline resistance to        infected or transfected host bacteria.    -   2. The tet fragment introduces unique restriction sites that can        be exploited for cloning foreign DNA sequences.

Thus, fd-tet was used, for example, as a cloning vector by exploitingthe unique Hind III site, situated in the tet fragment, to clone HindIII digested phage X DNA (Zacher et al, 1980). Furthermore, Smith hasreported the introduction of a second pVIII gene in the same regiongenerating a “type 88” vector designated f88.4, and the successfulproduction of pVIII mosaic phages (Zhong et al, 1994).

However, a point for concern is the report that foreign DNA, cloned intothe intergenic region of filamentous phages, tends to be unstable (Modelet al, 1988; Sambrook et al, 1989; Vieira et al, 1987). Occasionally,parts of the cloned DNA are deleted and the rate of the deletion eventtends to be directly correlated with the size of the inserted fragment.Therefore, the question of the genetic stability of DNA inserts clonedinto the intergenic region was examined.

Genetic Instability in fd-tet Vector

Purified fd-tet dsDNA generated two distinct electrophoretic patterns onagarose gels when isolated from two different E. coli strains (K802 andK91KAN). The DNA isolated from K802 exhibited the supercoiled andrelaxed forms of the fd-tet genome (FIG. 2A, lane 1) whereas the DNAisolated from K91KAN contained the supercoiled and relaxed forms of twodistinct DNAs (FIG. 2A, lane 2). One DNA corresponded to the fd-tetgenome while the second compared well with the parent fd genome (FIG.2A, lane 3). The two purified fd-tet preparations and fd were thendigested with the Msc I restriction enzyme that should generate fulllength linear DNA for both fd and fd-tet (the Msc I restriction site isunique in both fd and fd-tet, see FIG. 1). Digestion of fd-tet from K802and fd each generated only one band with the expected linear sizes of9183 bp and 6414 bp respectively (FIG. 2B, lanes 1 and 3). However,digestion of fd-tet derived from K91KAN generated two bands, oneindistinguishable in mobility from the linear fd-tet and the secondcorresponding in size to linear fd (FIG. 2B, lane 2). This indicatesthat for a substantial amount of DNA, a deletion of approximately 2.8 kboccurred when the fd-tet genome was produced in K91KAN bacteria.

Two points, therefore, must be addressed:

-   -   1. What DNA sequences are lost through the deletion process?    -   2. How are the differences in the bacterial strains related to        the apparent genetic instability?        Deletion of the tet Fragment

As the deleted DNA was 2.8 kbp, it was postulated that it mightcorrespond to the tet-fragment (2775 bp) residing in the intergenicspace of fd-tet (see FIG. 1). Therefore, the SnaB I/Ava I digests offd-tet DNA derived from both bacterial strains as before were compared.The SnaB I site is unique in fd, however, it appears twice in fd-tetwhile the Ava I site exists only in the tet fragment of fd-tet. The SnaBI/Ava I double digestion of fd-tet isolated from K802 generated threefragments with the expected sizes: 4381 bp, 2690 bp and 2112 bp (FIG. 3,lane 1). The double digestion of the K91KAN derived fd-tet DNA, however,generated four DNA fragments (FIG. 3, lane 2); the three fragmentscorresponding to fd-tet and an additional fragment corresponding to thedeletion product similar in size to a single cut full length 6414 bp fd(FIG. 3, lane 3, compare also with FIG. 2B). This indicates that theSnaB I-Ava I fragment of the tet insert in the intergenic region wasdeleted from fd-tet.

This conclusion was further supported by BspH I/Hind III digestion offd-tet derived from both bacteria. The Hind III site is unique to fd-tet(see FIG. 1). The BspH I site appears only once in fd and twice infd-tet (see FIG. 1, the second BspH I site is situated in the tetfragment). The BspH I/Hind III digestion of fd-tet DNA isolated fromK802 generated the expected three fragments of 4676 bp, 2366 bp, and2141 bp (FIG. 3, lane 4). However, the BspH I/Hind III double digestionof fd-tet DNA isolated from K91KAN generated not only the three fd-tetderived fragments but also a fourth fragment (approximately 6.4 kb)corresponding in size to single cut full-length fd (FIG. 3, lane 5, notearrow) derived from the deletion product. This not only confirms theselective deletion of the tet fragment but extends the boundaries of thedeletion beyond the SnaB I site to at least the BspH I site (see FIG.1).

Is the deletion a precise reversion to fd? If so, one would reconstitutethe original BamH I site used to clone the tet fragment, and thusgenerate two restriction fragments (3425 bp and 2989 bp as in the fdBamH I digest, see FIG. 4A lane 3) at the expense of the linearizeddeletion product (see for example the Msc I digest in FIG. 2B lane 2).As can be seen in FIG. 4A (lane 2), BamH I digestion of fd-tet isolatedfrom K91KAN produced a pattern similar to that shown for Msc I digest.Two bands corresponding to the linearized fd-tet (9183 bp) and thefull-length linearized deletion product (approximately 6.4 kb) weregenerated. This thus illustrates that although the deletion encompassesmuch or all of the tet fragment, it does not precisely regenerate thesecond BamH I site of the fd parent molecule.

Thus, the next question addressed, once the 2.8 kbp deletion was mappedto the vicinity of the tet fragment, was whether or not the excisionsites lay within or outside of the tet fragment. For this the BstY Idigest illustrated in FIG. 4B was performed (two BstY I sites, createdduring the fd-tet construction, precisely bracket the tet fragment, seeFIG. 1). The gel pattern obtained for the BstY I digest of fd-tetderived from K802 had three fragments (3425 bp, 2983 bp and 2775 bp)identical in size to those generated when K91KAN derived DNA was used(FIG. 4B, compare lane 1 with lane 2). This demonstrates that at leastone of the two BstY I sites was retained in the deleted product.Therefore, sequences immediately adjacent to the original BamH I site,must also be preserved in the deleted fd-tet, a fact that may berelevant to the mechanism of the deletion process (see Discussion).

The Deletion is recA Independent and F+Dependent

K802 and K91KAN strains of E. coli were introduced by Smith to produceand use the phage display vectors he has pioneered (Parmley, 1988). Theprinciple consideration is that phage infectability is dependent on theF-factor. Therefore, manipulation of the DNA vector, its modificationsand construction can be made in the absence of infection in F-bacteria(such as K802), thus avoiding multiply infected bacteria. Once thevector is completed, then in order to screen a phage library or toamplify and generate high titers of phages one must use F⁺ bacteria(e.g., K91KAN).

Table 1 illustrates that in a variety of E. coli strains tested, the tetdeletions were detected only in the F⁺ strains. Most noteworthy is thecomparison between the isogenic DH5α and DH5α F′ E. coli strains. Heretoo the deletion occurs exclusively in the DH5αF′ strain. These strainsare identical in their genotypes except that DH5αF′ carries theF-factor. This indicates that the deletion is F-factor dependent.Moreover, both strains are RecA⁻ in which the recA gene is mutated andthus classical homologous recombination is not possible. Therefore, thedeletion event is recA independent. TABLE 1 The Deletion is F⁺ Dependentand recA Independent E. coli Strain redA F-episome Deletion K802 + − −K91KAN + + + DH5α^(a) − − − DH5αF′^(a) − + + JM109 − + + XL1-Blue − + +MC1061^(b) + − − NM554^(b) − − −^(a)DH5α and DH5αF′ are isogenic strains; DH5αF′ contains the F-episome^(b)MC1061 and NM554 are isogenic strains; NM554 is recA⁻

As the F-factor is essential when using filamentous phages, working withF⁺ bacterial strains cannot be avoided. Therefore, introducing therecombinant pVIII gene in a region prone to F⁺ dependent deletions ispotentially problematic. The suitability of the non-coding regionbetween the wild type pVIII gene and the pIII gene as a site for geneticmanipulation and introduction of a second pVIII gene was thereforeexplored.

Construction of the ftac88 Vector

Designing a phage display vector with the aim of introducing a secondpVIII gene in the non-coding region between the wild type pVIII and pIIIgenes presents two obstacles that must be considered.

-   -   1. Unique restriction sites for cloning are unavailable in this        region.    -   2. This region contains an essential overlapping promoter and        rho-independent transcription terminator that are inseparable        (see FIG. 5;    -   see also Van Wezenbeek et al, 1980).

The terminator terminates the transcription of upstream genes endingwith the pVIII gene, whereas the promoter initiates the transcription ofdownstream genes starting with the pIII gene. Therefore, the strategyadopted to insert the recombinant second pVIII gene was to duplicatethis promoter/terminator as well. Ultimately, a “type 88” expressionvector (designated ftac88) was constructed as follows:

The first step was to generate the modified pVIII gene. This wasdesigned to contain unique restriction sites that could be used tointroduce foreign DNA sequences and thus enable the presentation oftheir corresponding encoded peptides at the N-terminus of the pVIIIprotein. Such a modified pVIII gene, designated pVIIISTS, was producedby “SOEing” PCR (Horton et al, 1990) mutagenesis in which the GAC codonfor residue Asp, at position 4 of mature wild type pVIII protein, wasreplaced by an insert of 62 base pairs (“STS” insert, FIG. 6A). Thisinsert contained two Sfi I sites that bracketed two translationstop-codons followed by the trpA transcription terminator. Thus, thetranscription of the pVIIISTS gene terminates prematurely, rendering theexpression of this gene silent. In order to express this pVIII gene onemust exchange the Sfi I flanked stuffer for a functional in-frame insertof DNA. The PCR was designed to generate a fragment corresponding to theSnaB I-BamH I segment of fd-tet (see FIG. 5) that was cloned into thepGEM-T vector, designated pGEM-T(p8STS) (FIG. 6B).

Next, a wild type pVIII gene was cloned into the SnaB I site ofpGEM-T(p8STS). This was achieved by PCR of the wild type pVIII genesegment from fd-tet starting from the upstream SnaB I site andcontinuing just beyond the overlapping promoter/terminator. Thedownstream antisense primer incorporated three unique restriction sitesas well. The PCR product was cloned into the blunt ends of SnaB I cutpGEM-T(p8STS) thus generating the desired pGEM-T(p88STS). As isillustrated in FIG. 6C the SnaB I-BamH I fragment of pGEM-T(p88STS)contains two pVIII genes, duplicated promoter/terminator elements andseveral unique cloning sites.

To complete the generation of the “type 88” phage display vector allthat was required was to replace the SnaB I-BamH I segment of fd-tetwith the corresponding SnaB I-BamH I fragment of pGEM-T(p88STS).However, fd-tet contains two SnaB I sites (see FIG. 5). Therefore, theSnaB I site in the tetR gene of fd-tet was removed. For this, fd-tetgenome was digested with Xba I/BstX I to remove the 725 bp fragmentcontaining the SnaB I site that was replaced by a linker containing anAva II site. This intermediate modified fd-tet was then digested withSnaB I/BamH I and the wild type segment was exchanged for thecorrespondingly cut fragment derived from pGEM-T(p88STS). Furthermore, atac promoter was introduced between the Xho I and Sac I sites to producethe “type 88” vector designated ftac88 shown in FIG. 7.

It was then necessary to evaluate whether or not the ftac88 vector canproduce mosaic phages displaying both wild type and chimeric pVIIIproteins. For this a linker encoding the linear peptide APAGFAIL (SEQ IDNO:28) (the epitope corresponding to the anti-gp120 mAb GV4H3 (Denisovaet al, 1996)) was inserted in frame between the two Sfi I sites,eliminating the trpA terminator. As demonstrated by the dot blotanalysis (FIG. 8), this construct (ftac88(4H3)) produced phages thatwere recognized by the GV4H3 mAb.

Genetic Instability in ftac88

The orientation of the wild type pVIII and the modified pVIIISTS genesin the ftac88 vector is a direct tandem repeat. The construction of thisvector was performed in recA⁻ bacteria and as such excludes thepossibility of recA dependent homologous recombination. However, in viewof the fact that the deletion of the tet fragment in fd-tet (describedabove) is recA independent and F⁺ dependent, the genetic stability ofthe modified pVIIISTS gene in DH5αF′ was examined. For this, ftac88 DNApreparations from DH5α and DH5αF′ bacteria were compared.

Double digestion of ftac88 with SnaB I and BamH I should release a 1378bp fragment containing both pVIII genes (see FIG. 7). Indeed, SnaBI/BamH I double digestion produced only the expected 1378 bp fragment,regardless of the source of the DNA (FIG. 9, lane 1, 2). As expected,the deletion of the tet fragment still occurred in DH5αF′ (FIG. 9, lane2; indicated by the upper arrow). Thus one can conclude that themodified pVIIISTS gene is genetically stable in ftac88.

However, the purpose of the ftac88 vector is to display peptides aschimeric pVIII proteins and as such, it was important to furtherinvestigate the genetic stability of the modified pVIIISTS gene when itcontained a peptide coding sequence rather than the stuffer with thetrpA terminator. For this, the ftac88 DNA construct expressing the GV4H3epitope (above) was prepared from DH5α and DH5αF′ bacteria and used forthe double digestion, SnaB I/BamH I analysis, as above. Here thesituation was surprising. Digestion of the DNA derived from DH5αreleased as expected a 1376 bp fragment (FIG. 9, lane 3). However, SnaBI/BamH I digestion of the same DNA prepared from DH5αF′ generated anadditional smaller fragment of approximately 950 bp (FIG. 9, lane 4).This indicates that a deletion of approximately 400 bp occurred. Thisdeletion corresponded to selective loss of the recombinant pVIII geneand reconstitution of the wild type pVIII-pIII configuration asconfirmed by sequence analysis (not shown). Therefore, it was concludedthat the modified pVIIISTS gene, when containing coding sequences,becomes genetically unstable in DH5αF′. This also suggests that thestability of the ftac88 observed in DH5αF′ is rendered due to the trpAterminator which could imply that the deletion process may requiretranscribed regions.

In summary, it is clear that the non-coding region between pVIII andpIII can be used. Moreover, the obstacle of the overlappingpromoter/terminator can be overcome. However, genetic instability of therecombinant pVIII gene was still a problem.

Construction of fth1 Vector

In light of the above experience in designing, constructing and testingthe ftac88 vector, it was decided to redesign a novel “type 88” vectorthat would overcome the problems of genetic instability and includeother improvements. The attributes of such a vector, designated fth1(FIG. 10) are listed herewith:

-   -   3. The second pVIII gene contained the 62 bp “STS” insert as in        ftac88. However, the gene was constructed using synthetic        oligonucleotides to generate a pVIII protein of identical amino        acid sequence yet coded for with alternative codon usage. This        obviated the formation of tandem repeats, a configuration that        is prone to deletion events as illustrated in the analysis of        ftac88 (above). The homologous gene was designated pVIIISTSh.        The pVIIISTSh gene was cloned, as in ftac88, between the wild        type pVIII and pIII genes    -   4. To avoid the generation of additional tandem repeats, an        alternative HP terminator (Nohno et al, 1986) was introduced        into the 3′ region of the wild type pVIII gene ten codons        upstream to the pVIII stop codon. Moreover, the severed 3′        region coding for the C-terminal domain of the wild type pVIII        protein was reconstituted using alternative codon usage (so to        reduce homology within the −35 box of the promoter of the pIII        gene residing in this region (Van Wezenbeek et al, 1980)).    -   5. The tetracycline resistance gene was cloned between the two        pVIII genes. This was accomplished via PCR of the open reading        frame of the tetracycline resistance gene only and placing it        downstream to a kanamycin promoter (derived from M13K07 phage).        This was done so as to avoid using the bi-directional        tetracycline promoter (see FIG. 5) which would otherwise        generate an anti-sense transcript of the wild type pVIII gene        were it used in this new position. Also the exclusion of the        tetracycline repressor is advantageous as the tetracycline        resistance gene thus becomes constitutively expressed, making        pre-incubation with low levels of tetracycline to induce        resistance unnecessary.    -   6. The original intergenic space containing the ori sequences        was reconstituted. Previously, the tet fragment used by Smith to        construct fd-tet, was inserted into the fd ori(−) rendering this        vector and its derivatives low copy number and thus producing        relatively low phage titers (Smith, 1988). Reconstituting the        intergenic region in fth1 provided two advantages: the copy        number of the DNA was greatly increased and the titers of        secreted phage increased as well (see below).

The construction of fth1 (see FIG. 11) was accomplished using “SOEing”PCR as described above enabling the introduction of multiple cloningsites and novel genes into the non-coding region between the wild typepVIII and pIII genes of fd-tet.

The first step was to modify fd-tet by the introduction of a shortsegment of DNA, ten codons upstream from the stop codon of the wild typepVIII gene (FIG. 11B). This segment not only provided novel restrictionsites but also separated the promoter/terminator adjacent to the pIIIgene from the wild type pVIII gene by reconstituting the severed lastten codons of the pVIII open reading frame (intermediate vector 1 (IV-1)in FIG. 11B).

Subsequently, additional intermediate vectors were produced (IV-2 andIV-3, see Materials and Methods) which eventually led to IV-4 (FIG. 1C).In the latter, the novel HP terminator was incorporated downstream tothe reconstituted wild type pVIII gene followed by the tetracyclineresistance open reading frame driven by the Kanamycin promoter.Moreover, the ori(−) in the intergenic region was also reconstituted inIV-4.

The final vector was produced by cloning the synthetic pVIIISTSh gene,driven by the tac promoter, into the Bgl II/Eag I digested IV-4 vectorto give fth1 (see detailed map, FIG. 10).

The questions to be evaluated regarding the utility and advantages offth1 therefore were:

-   -   1. Was the vector, and particularly the tetracycline resistance        and recombinant pVIII genes, genetically stable?    -   2. Could the recombinant pVIII gene be used to stably express        peptide inserts?    -   3. Was the fth1 a high copy number vector?    -   4. Could high titers of fth1 progeny be achieved?

In order to answer these questions, the GV4H3 epitope was introducedbetween the Sfi I sites in fth1 (as done previously for ftac88). Thisconstruct, called fth1(4H3), produced mosaic phages presenting the GV4H3epitope that were recognized by the GV4H3 mAb (FIG. 8).

The stability of the tetracycline resistance gene and the modifiedpVIIISTSh gene was tested using restriction analyses as before.Digestion of fth1 and fth1(4H3) DNA, derived from DH5α or DH5αF′, withBstN I generated identical fragments as is illustrated in FIG. 12. BstNI sites appear twice in both fd (see FIG. 1) and fth1 (see FIG. 10).BstN I digestion of fd generated the expected two fragments of 5456 bpand 952 bp sizes (FIG. 12, lane 1). The latter contains the non-codingregion between pVIII and pIII genes. As this region in fth1 contains thetetracycline resistance and the recombinant pVIIISTSh genes, the 952 bpfragment was shifted to generate the 2777 bp and 2775 bp bands in BstN Idigests of fth1 and fth1(4H3) respectively derived from both DH5α andDH5αF′ (lane 2-5). Any deletions of either the tetracycline resistancegene or the recombinant pVIII gene or their parts would generate DNAfragments ranging in size from 952 bp to 2777 bp. No such fragmentscould be detected illustrating that the fth1 vector is geneticallystable.

The functionality of the reconstituted ori(−) is directly apparent. Inseveral independent purifications of fth1 DNA, 0.5-1 mg DNA/liter ofbacteria was routinely obtained, as opposed to the typical yields of 50μg DNA per liter of ftac88 harboring bacteria. Similarly a hundred foldincrease is typically seen in phage titers for which 1012 phages/ml ismeasured for fth1 as compared to 10¹⁰ phages/ml achieved for fd-tet andits derivatives.

Thus the fth1 vector provides a convenient and stable “type 88” phagedisplay expression system.

Discussion

The selective deletion of the tet fragment from the intergenic space hasbeen critically evaluated as a first step towards producing a novel“type 88” phage display vector. The present inventors have observed thatin F⁺ bacterial strains a 2.8 kb fragment of DNA is excised from theregion of the foreign tet insert, Smith originally introduced into theB-stem/loop of the ori(−) structure (Zacher et al, 1980). The loss ofthe insert's Ava I site, which overlaps the original remnant BamH Isite, illustrates that downstream aspects of the insert are notpreserved in the deleted form. The loss of the BspH I site and all fourof the insert's Hinc II sites (not shown) argues that no more than 18base pairs of the insert can be retained at the other extreme.Preservation of at least one BstY I site, yet lack of reconstitution ofthe original BamH I site, further stresses the close but not precisecorrespondence of the deleted fragment with the original tet insert.

As the insert is flanked by the inverted repeats that create theB-stem/loop and that these repeats are retained in the deleted form ofthe phage, one might postulate that the mechanism generating thedeletion involves homology and base paring. Clearly, however, this cannot be homologous recombination as that would require direct repeats ofhomologous stretches of over 50 bases and of course, a functional RecAprotein in order to enable a deletion event (Clark, 1973; Matfield etal, 1985). The deletion events in our experiments occurred in recA⁻bacterial strains. An alternative mechanism, the “slippage mechanism”proposed by Lovett (Feschenko et al, 1998), also requires direct repeatsand therefore can not explain the deletion. To the best of ourknowledge, no known mechanism can explain the deletion of the tetfragment as observed.

One might imagine that during the course of replication, the invertedrepeats, flanking the tet fragment, align to form a B-stem/loop ofsorts, greatly distorted by a grossly extended “bubble” (where the BamHI site had been), consisting of the 2.8 kb insert. During replication,the polymerase might be able to “skip” from one side of the bubble tothe other thus omitting the entire insert and generating the deletion.Assuming that this is a very rare event one must still address the factthat in K91Kan cells over 50% of the DNA isolated was of the deletedform. Moreover, why is the deletion F⁺ dependent?

The matter of copy number might be explained due to two selectiveadvantages that are realized in the deleted form. The mere fact that thegenome is 2.8 kb shorter than fd-tet, should speed up the replication.More important may be the fact that the deletion most probably partiallyreconstitutes the ori(−) structure. The ramifications of a markedly morefunctional ori(−) would substantially enable more effective productionof double stranded DNA.

Another possibility may be related to the packaging of phages forsecretion. The A-stem/loop structure, situated adjacent to theB-stem/loop, is the packaging-signal of the phage (Van Wezenbeek et al,1980). It stands to reason that the tet insert may sterically hinder thepackaging of the recombinant phages, a burden that would be removed inthe deleted form.

However, the major phenomenon encountered here is the fact that thedeletion is detectable only in the F⁺ bacteria. This might be due to thefact that even rare events can be markedly amplified when superinfection of bacteria can take place. Obviously, for such superinfection, the bacteria must be compatible, a situation only satisfiedin F⁺ bacteria. Therefore, it is proposed that the deletion leads toshorter and more efficient replicating forms of the phage, albeit a rareevent. Subsequently, however, such deleted phages can be markedlyamplified in F⁺ bacteria via super infection thus enabling accumulationof very substantial amounts of the deleted form. However, thepossibility cannot be excluded that unknown proteins encoded by theF-episome might contribute to the deletion events either by directlyenhancing the process or indirectly inducing the expression of bacterialproteins participating in the process.

In view of the above considerations, it was concluded that theintergenic region of the phage is not the optimal site for introductionof a second pVIII gene. Not that this has not led to an effective “type88” vector in the past such as the f88.4 vector constructed by Smith(Zhong et al, 1994). In the f88.4 vector, the recombinant pVIII gene wasintroduced into the tet fragment of fd-tet. However, one would expectwith such a vector the gradual appearance of deleted forms of the phagesand loss of their peptide inserts displayed by the chimeric pVIIIprotein. Indeed, such “contaminating”, fast-growing, tetracyclinesensitive phages have been reported by Scott when they used the f88.4vector (Bonnycastle et al, 1996).

An alternative cloning site for the second pVIII gene was tested, namelythe non-coding region between the pVIII and pIII genes. The ftac88described in this report illustrated that such a construction is notonly possible but able to produce workable amounts of recombinantphages. However, once again an unexpected obstacle was encountered assubstantial amounts of deletions of the recombinant pVIII were observed.This deletion process also occurred in a recA⁻ background, excludingtherefore the possibility of classical homologous recombination. Incontrast to the situation of the deletion of the tet insert however, thedeletion of the modified pVIII gene entails reasonably long directrepeats. Thus, whereas homologous recombination is not an option, theslippage mechanism which is recA independent, described by Feschenko andLovett (Feschenko et al, 1998), might be responsible for the selectiveloss of the second pVIII gene in ftac88. This would entail the slippedmisalignment of the nascent strand during DNA replication. Duringreplication of the repeated DNA the nascent strand becomes displacedfrom its template and pairs with the other copy on the template strand.Then, replication is resumed and the slipped misalignment can lead toeither deletion or expansion within tandem repeat arrays. Therefore,deletions due to the slippage mechanism are homology dependent, arequirement satisfied in ftac88.

The selective loss of the recombinant pVIII gene further supports theslippage mechanism. This mechanism requires the stabilization of theslipped misalignment structure (Feschenko et al, 1998; Lovett et al,1996). The longer the displaced nascent strand, the more stabilized theslipped misalignment structure becomes. The recombinant pVIII genecontains a foreign sequence in its 5′ region. This severs the repeatinto two parts, a short sequence upstream and a long downstreamsequence. The long sequence, therefore, enables the stabilization of theslipped misaligned structure more efficiently than the short one.Therefore, excision sites in the long downstream identical sequenceshould predominate thus eliminating upstream sequences including theforeign DNA insert.

The actual deletion event may be a very rare one, typical of theslippage mechanism. One can postulate that deleted DNA products could beamplified due to F⁺ dependent superinfection (see discussion above).

Therefore, the last aspect that must be considered is why the pVIIISTSform is stable as opposed to those constructs in which the stuffercontaining the trpA terminator is exchanged for a peptidecoding-sequence (such as the GV4H3 epitope). The stability rendered bythe presence of the trpA transcriptional terminator indicates thattranscription might be a positive and necessary factor. The trpAterminator stops the transcription of the recombinant pVIII geneprematurely and consequently no deletion is observed. Transcriptionthrough the second homologous DNA repeat appears to be a necessarypre-requisite that provides the opportunity to form the misalignedstructure that leads to subsequent deletion during DNA replication. Sucha mechanism may explain how misalignment can exist between the first andsecond repeats at the moment when the first repeat is being replicatedyet the second downstream repeat is still in the closed double strandedDNA configuration. In order to mispair the nascent strand to the closeddouble-stranded repeat, the latter would have to be opened. In theabsence of RecA protein, which catalyzes such reactions (West, 1992),transcription might be an efficient mode for opening double strandedDNA. However, as opening double stranded DNA during transcription istransient, this mode would be efficient only when the DNA repeats are inreasonably close proximity. Indeed, the deletion frequency, reported forthe slippage mechanism, is correlated directly with the distanceseparating the DNA repeats (Lovett et al, 1994; Bi et al, 1994).Moreover, this mechanism would require that the wild type pVIII gene bethe first to be replicated. Indeed, the most extensive replicationduring infection is that of the + strand, proceeding from the intergenicregion through the wild type pVIII gene to the recombinant pVIII gene(Horiuchi et al, 1976).

As a result of the above analyses and the experiences gained through theconstruction and characterization of the ftac88 vector, an improvednovel vector that corrects for the problems encountered was ultimatelydesigned. This vector has been designated fth1 and has the followingadvantages.

-   -   1. The tetracycline resistance and the recombinant pVIII genes        were introduced between the wild type pVIII and pIII genes.    -   2. To avoid the presence of repetitive sequences of any kind,        the two transcription units of the phage genes were separated by        inserting a novel HP-terminator. Furthermore, the wild type        pVIII C-terminal domain encoding region was reconstituted using        alternative codon usage. The recombinant pVIII gene was        generated using alternative codon usage as well. These        attributes render the fth1 vector genetically stable.    -   3. The fth1 vector contains the wild type intergenic region.        Thus, the minus-strand DNA synthesis occurs as efficiently as in        wild type fd rendering fth1 a high copy number vector. This        enables one to isolate and purify consistently large amounts of        the double-stranded DNA from fth1 harboring bacteria (500 μg per        liter as oppose to 50 μg of ftac88). Furthermore, fth1 produces        high phage titers −10¹² phages per ml as oppose to 10¹⁰ phages        per ml typical for fd-tet and its derivatives.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention. Thusthe expressions “means to . . . ” and “means for . . . ”, or any methodstep language, as may be found in the specification above and/or in theclaims below, followed by a functional statement, are intended to defineand cover whatever structural, physical, chemical or electrical elementor structure, or whatever method step, which may now or in the futureexist which carries out the recited function, whether or not preciselyequivalent to the embodiment or embodiments disclosed in thespecification above, i.e., other means or steps for carrying out thesame functions can be used; and it is intended that such expressions begiven their broadest interpretation.

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1. A DNA molecule comprising the DNA of a filamentous phage into which has been inserted a recombinant DNA sequence comprising a multiple cloning site comprising a series of restriction sites that do not otherwise appear in said DNA of the filamentous phage, said recombinant DNA sequence being designed and inserted such that the multiple cloning site is placed between a terminator for the wild type pVIII gene and an initiator for the wild type pIII gene.
 2. A DNA molecule in accordance with claim 1, wherein a positive selection marker is present in the multiple cloning site.
 3. A DNA molecule in accordance with claim 1, wherein an additional bacteriophage gene is present in the multiple cloning site.
 4. A DNA molecule in accordance with claim 1, wherein said additional bacteriophage gene is a second pVIII gene.
 5. A DNA molecule in accordance with claim 1, wherein said additional bacteriophage gene is a second pIII gene.
 6. A DNA molecule in accordance with claim 1, wherein said recombinant DNA sequence is designed and inserted such that a DNA sequence encoding a foreign peptide is incorporated into the pIII gene at the N-terminal region thereof.
 7. A DNA molecule in accordance with claim 1, wherein said multiple cloning site placed between said wild type pVIII gene (the first pVIII gene) and the wild type pIII gene comprises DNA encoding an additional pVIII protein (the second pVIII gene) and DNA encoding a polypeptide of interest other than pVIII.
 8. A DNA molecule in accordance with claim 7, wherein the DNA encoding the second pVIII gene uses alternative codons which are other than the native codons for encoding one or more of the amino acid residues of the pVIII protein, such that no stretch of more than 30 bases of the native sequence appear therein.
 9. A DNA molecule in accordance with claim 8, wherein no stretch of more than 20 bases of the native sequence appear in the DNA encoding the second pVIII gene.
 10. A DNA molecule in accordance with claim 7, wherein the native pVIII DNA sequence is separated from the recombinant pVIII DNA sequence by a terminator which is not native to the filamentous phage DNA.
 11. A DNA molecule in accordance with claim 7, wherein DNA encoding the native amino acid sequence of the pVIII C-terminal/pIII −35 box is maintained at the region immediately upstream to the initiation of the pIII gene.
 12. A DNA molecule in accordance with claim 11, wherein said region encoding the pVIII C-terminal domain in said C-terminal/−35 box uses alternative codons which are other than the native codons, such that no stretch of more than 30 bases of the native sequence appear therein.
 13. A DNA molecule in accordance with claim 12, wherein no stretch of more than 20 bases of the native sequence appear in the region encoding the pVIII C-terminal/−35 box.
 14. A DNA molecule in accordance with claim 7, wherein a positive selection marker is inserted between the native and recombinant pVIII genes.
 15. A DNA molecule in accordance with claim 14, wherein said positive selection marker is the tetracycline resistance gene.
 16. A DNA molecule in accordance with claim 15, wherein a unidirectional promoter is substituted for the native bidirectional tetracycline resistance gene promoter.
 17. A DNA molecule in accordance with claim 7, wherein the native intergenic space region is present.
 18. A DNA molecule in accordance with claim 7, wherein a promoter is used for the second pVIII gene and DNA encoding the peptide of interest, which promoter is not native to the native filamentous phage.
 19. A DNA molecule in accordance with claim 7, wherein said recombinant DNA sequence is designed and inserted such that a DNA sequence encoding a second peptide of interest is incorporated into the pIII gene at the N-terminal region thereof.
 20. A filamentous phage encoded by the DNA of claim
 7. 21. A DNA molecule in accordance with claim 7 wherein said polypeptide of interest is a random peptide.
 22. A library of filamentous phages encoded by a DNA molecule in accordance with claim 21, wherein said library comprises a plurality of phages each having a different random peptide as the polypeptide of interest.
 23. A library in accordance with claim 22, wherein the polypeptides of interest in said plurality of phages comprise all possible random peptides of a given length or a fraction thereof which maintains the complexity thereof.
 24. A filamentous phage encoded by a DNA molecule in accordance with claim 7, wherein said polypeptide of interest is a peptide which comprises a contiguous fragment of the sequence of an antigen of interest.
 25. A library of filamentous phages in accordance with claim 24, wherein said library comprises a plurality of phages, said peptide in each of said plurality of phages comprising a different contiguous fragment of the sequence of the antigen of interest.
 26. A library in accordance with claim 25, wherein the polypeptides of interest in said plurality of phages comprise all possible overlapping contiguous fragments of the antigen of interest.
 27. A method of screening for a molecule which binds to a peptide of interest comprising bringing a molecule to be screened into contact with a filamentous phage in accordance with claim 20 which expresses said peptide of interest and, if said molecule binds to said peptide of interest, identifying and producing said molecule.
 28. A method of screening for peptides which bind to a molecule of interest comprising bringing the molecule of interest into contact with a library of filamentous phages in accordance with claim 22; identifying any peptide expressed by a phage which binds to said molecule; and producing a polypeptide which comprises said peptide.
 29. A method of screening for peptides which bind to a molecule of interest comprising bringing the molecule of interest into contact with a library of filamentous phages in accordance with claim 25; identifying any peptide expressed by a phage which binds to said molecule; and producing a polypeptide which comprises said peptide.
 30. A DNA molecule comprising the DNA of a filamentous phage, into which has been inserted between the wild type pVIII gene (the first pVIII gene) and the wild type pIII gene, recombinant DNA sequence encoding an additional pVIII protein (the second pVIII gene) and DNA containing a region of bases flanked by two identical restriction enzyme sites, which sites do not appear anywhere else in the DNA molecule.
 31. A DNA construct for insertion into the DNA of a filamentous phage, said construct comprising a multiple cloning site comprising a series of restriction sites that do not otherwise appear in the DNA of the phage into which the construct is intended to be inserted, said construct having one end compatible for ligation to a restriction enzyme site upstream of the pIII gene of the phage into which the construct is intended to be inserted and another end compatible for ligation to a restriction enzyme site downstream of the pVIII gene of the phage into which the construct is intended to be inserted.
 32. A DNA construct in accordance with claim 31 further including a positive selection marker.
 33. A DNA construct in accordance with claim 31 further including a second pVIII gene. 